A critical component of agriculture biotechnology is the use of highly regulated promoters to express agronomically important genes in crop plants so that genes of interest are expressed at optimal levels in appropriate tissues.
Monocots form a substantial portion of the world's food supply. Sugarcane is considered as one of the major worldwide crop for sugar supply with a net value of $20 billion per year. This crop could benefit from biotechnology approaches to engineer plants for disease, pest and herbicide resistance as well as improved sugar yield. However, compared to other major crops, there has been little research in developing sugarcane specific technologies such as genes and promoters that are functional in sugarcane. At the present time, there are no public-domain sugarcane promoters available for use in sugarcane transformation.
The selection of a promoter is often a critical factor in determining when and where within the plant the gene of interest is expressed.
A number of gene promoters that drive high levels of constitutive transgene expression are available for monocot plants. These include the maize ubiquitin promoter (Quail et al., 1996, U.S. Pat. No. 5,510,474), the rice actin1 promoter (McElroy and Wu, 1997, U.S. Pat. No. 5,641,876), various enhanced cauliflower mosaic virus (CaMV) 35S promoters (Mitsuhara et al., 1996, Plant Cell Physiol. 37:49-59) and promoters isolated from banana streak virus (Schenk et al., 2001, Plant Mol. Biol. 47:399-412). Promoters that have been isolated so far from sugarcane correspond to two polyubiquitin genes and confer constitutive gene expression in non-host plants such as rice (Wei et al., 1999, J. Plant Physiol. 155:513-519; Wei et al., 2003, J. Plant Physiol. 160:1241-1251).
Constitutive promoters may be suitable for the production of a desired protein in large quantities in all tissues of the plant throughout development. The energy requirements for high level constitutive production of the protein are often so great that other normal plant growth processes are compromised. For example, expression of a protein in a non-tissue specific manner as directed by a constitutively active promoter has often resulted in slow-growing or dwarfed plants. Even though providing constitutive expression of a gene is often desirable, it is also desirable in many instances to direct high expression of a gene to particular tissues and/or time of development in a plant. Tissue-specific promoters are capable of selectively expressing an introduced gene in desired tissues. Tissue-specific promoters may also be inducible, e.g. activated by internal or external agents such as phytohormones or defense inducing agents.
Restricting expression of the target protein to a particular tissue or organ or to specific events triggered when the plant is challenged with an external agent may be desirable to minimize possible toxic effects of some agronomic gene products and to optimize overall plant growth and production. Furthermore, it is increasingly clear that promoter function varies from species to species. Thus it is essential to have promoters which are expressed specifically in target tissues of specific plants in order to genetically engineer plants.
Several promoters are currently being used for tissue-specific, heterologous gene expression in monocots. For example, the promoter regions from genes coding for hydrolases (β-glucanase), cysteine protease inhibitors (cystatin-1) or glucosidases (α-glucosidase) have been used to direct germination-specific expression of a heterologous DNA sequence in transgenic barley cells and kernels (Wolf, 1992, Mol. Gen. Genet. 234:33-42; Jensen et al., 1996, Proc. Natl. Acad. Sci. USA 93:3487-3491; Mikkonen et al., 1996, Plant Mol. Biol. 31:239-254; Jensen et al., 1998, U.S. Pat. No. 5,712,112; Lok et al., 2002, U.S. Pat. No. 6,359,196). The promoter for the glutamine synthetase gene has been also used to drive tissue-specific expression within the developing kernels of transgenic maize plants (Muhitch et al., 2002, Plant Science 163:865-872). Xylem- and phloem-specific promoters that are active in rice have also been reported, including the rice tungro baciliform virus and peroxidase gene promoters (Yin et al., 1997, Plant Journal 12:1178-1188; Ito et al., 2000, Plant Science 155:85-100); however it is not clear whether these promoters are active in other monocots, such as maize, sorghum and sugarcane. Furthermore, none of the above reported promoters are stem-regulated, which may be significant for crops such as sugarcane where the stem holds a large portion of the commercial value of the plant.